RESUMO
A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630â nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1â µm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60â nm.
Assuntos
Microscopia/métodos , Pironina/química , Rodaminas/química , Corantes Fluorescentes/química , Células HeLa , HumanosRESUMO
Caged rhodamine dyes (Rhodaminesâ NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λâ¼750â nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330-350â nm (molar extinction coefficient (ε)≈10(4) M(-1) cm(-1)) with a band edge that extends to about λ=440â nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511-633 and 525-653â nm, respectively). The unmasked dyes are highly colored and fluorescent (ε=3-8×10(4) M(-1) cm(-1) and fluorescence quantum yields (Ï)=40-85% in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodaminesâ NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375-420â nm light or intense red light (λ=775â nm). Protein conjugates with optimal degrees of labeling (3-6) were prepared and uncaged with λ=405â nm light in aqueous buffer solutions (Ï=20-38%). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40% of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodaminesâ NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.
Assuntos
Compostos Aza/química , Corantes Fluorescentes/síntese química , Indanos/química , Rodaminas/química , Animais , Chlorocebus aethiops , Citoesqueleto/química , Corantes Fluorescentes/química , Microscopia de Fluorescência , Fotólise , Proteínas/química , Proteínas/metabolismo , Rodaminas/síntese química , Espectrometria de Fluorescência , Ácidos Sulfônicos/química , Raios Ultravioleta , Células VeroRESUMO
New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514â nm light; most of them emit light at λ=512-554â nm (compounds 1 q and 1r at λ=576 and 589â nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3â ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments.